Novel ligands and methods for preparing same

ABSTRACT

A process is disclosed for modifying a parent ligand by attaching to the parent ligand a conjugation agent that is reactive with a moiety of a target receptor to which the parent ligand binds such that a covalent bond is formable between the conjugation agent and the receptor moiety. Also disclosed are compositions, probes and methods of detecting and/or quantifying receptors using the modified ligands of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplication Ser. No. 60/178,756, filed Jan. 28, 2000, the disclosure ofwhich is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] This invention relates generally to ligand-receptor interactions.In particular, the present invention relates to modified ligands thatbind irreversibly to their cognate receptors, and to methods forpreparing such ligands. The novel ligands of the invention have utilityinter alia for investigating protein function and as drugs (inhealthcare, agricultural and environmental applications) for moreeffectively inhibiting or stimulating cognate receptor function.

BACKGROUND ART

[0003] Pharmacological receptors are intracellular or membrane-boundproteins which produce a pharmacological effect after binding with aspecific ligand. In this regard, a pharmacological receptor has a dualfunction to (a) detect a ligand signal by forming a ligand-receptorcomplex and to (b) conduct and translate the signal leading to thepharmacological effect.

[0004] Drugs can replace endogenous physiological ligands to interactwith receptors. A prerequisite for such a drug-receptor interaction isthe formation of a drug-receptor complex, just as in the case ofligand-receptor interaction. In contrast to physiological ligands thatstimulate an effect after binding to a receptor (receptor-mediatedeffects), drugs can be classified as (a) agonists or drugs whichstimulate an effect after binding to the receptor, and (b) antagonistsor drugs which do not stimulate an effect after receptor binding.

[0005] Several types of molecular interactions are possible fordrug-receptor binding including ionic bonds, hydrogen bonds, andhydrophobic bonds by van der Waals forces. The vast majority of receptorinteractions involve several kinds of binding simultaneously. Ionicbonds are important for the primary phase of drug-receptor interactionsince these bonds have the greatest or longest range. After the initialinteraction, fine-tuning takes place involving dipole-dipole-bonds,hydrogen bonds and hydrophobic bonds. Although all these interactionsalso fix the drug molecule in the receptor's active site, the bindingsare nevertheless reversible, as the force of interaction is very weak.Hence the pharmacological effectiveness of any drug is often affected byits own concentration in the plasma, as a decrease in plasma drugconcentration will increase the dissociation of drug molecule from itsreceptor.

[0006] Several agents are known to inhibit enzymes irreversibly orpseudoirreversibly and the precise mechanism of inhibition gives rise tosubtle differences in the inhibition profiles and the duration ofinhibition.

[0007] Many inhibitors of acetylcholinesterase react covalently withthis enzyme to form an acyl enzyme that deacylates more slowly than theacetyl enzyme formed with the natural substrate acetylcholine. Theacetyl enzyme forms rapidly by attack of the active site serine on thesubstrate. Transfer of the acyl group to the enzyme occurs through atetrahedral intermediate. The acetyl enzyme is rapidly hydrolyzed, witha half time of 10 μsec. These rapid acylation and deacylation steps giverise to a turnover rate of 10⁵ substrate molecules per enzyme moleculeper second. Cholinesterase inhibitors such as physostigmine andneostiginine form methylaminocarbamyol and dimethylaminocarbamoylenzymes, which have half times for deacylation of several minutes. Thus,by providing the enzyme with an alternative substrate, catalysis ofacetylcholine is precluded during the catalytic cycle for thecarbamoylating agent. The kinetic constants for the respective acylationsteps for the acetoxy and carbamoxy ester substrates do not greatlydiffer; hence the longer residence time of the carbamoyl enzymeconjugate is an important factor in favoring inhibition.

[0008] Several other enzymes are inhibited by covalent attachment of theinhibitor, giving rise to irreversibility. The hydrazines (phenelzine,isocarxazid metabolites) and the acetylenic agents (pargyline) areoxidized to reactive intermediates by monoamine oxidase. Theseintermediates attack the associated flavin cofactor on the enzyme Suchagents have been termed suicide substrates since their activationrequires catalysis by the very enzyme that they inactivate. Hence theinactivation process is mechanism-based. There are now many examples ofsuch substrates, activation of which by the enzyme results in covalentmodification of the enzyme or of an associated cofactor. Often thisoccurs by conjugation or association of the enzyme with its substratefollowed by a neighboring group attack. Several of the targets ofsuicide substrates have therapeutic significance. These include thepenicillinases and alanine racemases in antibacterial design; GABAtransaminase inhibitors for antiepileptic agents; lipoxygenase andcyclooxygenase inhibitors to control leukotriene and prostaglandinbiosynthesis, respectively; aromatase inhibitors to block formation ofestrogenic hormones; ornithine decarboxylase inhibitors as antiparasiticagents; and dopamine β-hydroxylase inhibitors to control catecholaminebiosynthesis. Many suicide substrates serve as antimetabolites and arepotential antineoplastic agents. The effectiveness of these inhibitorsdepends not only on their relative dissociation constants or K_(m)values compared with those of the endogenous substrate but also onkinetic competition between turnover of the suicide substrate and theinactivation event.

[0009] Omeprazole (PRILOSEC) is another well-known irreversible bindingdrug that has been released for clinical use. This drug inhibits gastricacid secretion by binding to the H⁺, K⁺-ATPase present only in theapical membrane of parietal cells. Omeprazole is especially useful inpatients with hypergastrinemia and may be valuable in those whose pepticulcer disease is not well controlled by H₂ antagonists. Omeprazolecontains a sulfinyl group in a bridge between substituted benzimidazoleand pyridine rings. At neutral pH, this drug is a chemically stable,lipid-soluble, weak base that is devoid of inhibitory activity. Thisneutral weak base reaches parietal cells from the blood and diffusesinto the secretory canaliculi, where the drug becomes protonated andthereby trapped. Protonated drug rearranges to form a sulfenic acid anda sulfenamide. The sulfenamide interacts covalently with sulfhydrylgroup at critical sites in the extracellular (luminal) domain of themembrane-spanning H⁺, K⁺-ATPase. Omeprazole must thus be considered asprodrug that needs to be activated to be effective.

[0010] Despite the availability of several agents that bind theirtargets irreversibly, there is a dearth of methods currently availablefor rationally designing ligands to irreversibly bind a target receptor.

DISCLOSURE OF THE INVENTION

[0011] The present invention arises, at least in part, from theunexpected discovery that by attaching a conjugation agent to a parentligand that reversibly binds a target receptor, wherein the conjugationagent is reactive with a moiety of the target receptor such that acovalent bond is formable between the conjugation agent and the moiety,the modified ligand thus produced is capable of binding the targetreceptor irreversibly Accordingly, in one aspect of the invention, thereis provided a process for modifying a parent ligand, comprisingattaching to said parent ligand a conjugation agent that is reactivewith a moiety of a target receptor to which said parent ligand bindssuch that a covalent bond is formable between said conjugation agent andsaid moiety.

[0012] Suitably, the conjugation agent is attached to the parent ligandthrough a spacer.

[0013] Preferably, the spacer is covalently attached to the parentligand.

[0014] Preferably, the spacer is covalently attached to the conjugationagent.

[0015] The spacer is suitably radical selected from the group consistingof alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, oxoalkyl,heterooxoalkyl, alkenyl, hetero alkenyl, aralkyl, hetero aralkyl, aryland heteroaryl radicals or any other molecular conformation which servesthe function of being a spacer. The length of the spacer arm is suitablyselected from a range of between about 0 Å and about 20 Å.

[0016] Preferably, the spacer is a non-hydrolysable radical underphysiological conditions.

[0017] Preferably, the conjugation agent is selected from the groupconsisting of a sulfhydryl group specific conjugation agent, an aminogroup specific conjugation agent, a carboxyl group specific conjugationagent, a tyrosine specific conjugation agent, an arginine specificconjugation agent, a histidine specific conjugation agent, a methioninespecific conjugation agent, a tryptophan specific conjugation agent, anda serine specific conjugation agent.

[0018] The sulfhydryl group specific conjugation agent may be selectedfrom the group consisting of N-maleimide, N-maleimide derivatives anddisulfide reagents including, but not restricted to,5′-dithiobis-(2-nitrobenzoic acid), 4,4′-dithiodipyridine,methyl-3-nitro-2-pyridyl disulfide, and methyl-2-pyridyl disulfide.

[0019] The amino group specific conjugation agent may be selected fromthe group consisting of alkylating agents including, but not restrictedto, α-haloacetyl compounds, aryl halides, aldehydes and ketones, andacylating agents including, but not restricted to, isocyanate,isothiocyanate, imidoesters, N-hydroxylsuccinimidyl ester, ρ-nitrophenylester, acyl chloride, and sulfonyl chloride.

[0020] The carboxyl group specific conjugation agent may be selectedfrom the group consisting of carbodiimides and carboxyl groupesterification reagents including, but not restricted to, diazoacetateesters and diazoacetamides.

[0021] The tyrosine specific conjugation agent may be selected fromdiazonium derivatives including, but not limited to, benzidine andbis-diazotized 3,3′-dimethylbenzidine.

[0022] The arginine specific conjugation agent may be selected from1,2-dicarbonyl reagents including, but not restricted to, glyoxal,phenylglyoxal, 2-3-butanedione and 1 ,2-cyclohexanedione.

[0023] The histidine specific conjugation agent is suitably selectedfrom the group consisting of alkylating agents including, but notrestricted to, α-haloacetyl compounds, aryl halides, aldehydes andketones, and acylating agents including, but not restricted to,diethylpyrocarbonate, ethoxyformic anhydride, isocyanate,isothiocyanate, imidoesters, N-hydroyxlsuccinimidyl ester, ρ-nitrophenylester, acyl chloride, and sulfonyl chloride.

[0024] The methionine specific conjugation agent may be selected fromthe group consisting of alkylating agents including, but not restrictedto, α-haloacetyl compounds, aryl halides, aldehydes and ketones.

[0025] The tryptophan specific conjugation agent may be selected fromthe group consisting of N-bromosuccinimide, 2-hydroxy-5-nitrobenzylbromide and ρ-nitrophenylsulfenyl chloride.

[0026] The serine specific conjugation agent may be selected from thegroup consisting of diisopropylfluorophosphate and acrylsulfonylfluorides including, but not restricted to,phenylmethyl-sulfonylfluoride.

[0027] The parent ligand may be any natural or non-natural ligand but ispreferably a biologically active ligand inclusive of known drugs andnaturally occurring or synthesized drug candidate compounds.

[0028] In another aspect, the invention provides a modified ligandproduced by the process broadly described above.

[0029] In yet another aspect of the invention, there is provided amodified ligand having the general formula:

L—R_(l)—I—A  (I)

[0030] wherein L is a parent ligand;

[0031] wherein A is a conjugation agent that is reactive with a moietyof a target receptor to which the parent ligand binds such that acovalent bond is formable between said conjugation agent and saidmoiety; and

[0032] R₁ is an optional spacer which preferably comprises anon-hydrolysable radical selected from the group consisting of alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, oxoalkyl, heterooxoalkyl,alkenyl, hetero alkenyl, aralkyl, hetero aralkyl, aryl and heteroarylradicals.

[0033] In a preferred embodiment, the modified ligand is interactivewith es nucleoside transporter and/ornucleoside/nucleotide/nucleobase-sensitive proteins, wherein saidmodified ligand has a general formula selected from the group consistingof:

[0034] wherein A is N-maleimide, 2-pyridyldithio, or halogen; X is NH,S, or O; Y is H, halogen, NH₂, or O; Z is H, halogen, or CH₃; R₁ isspacer arm comprising a non-hydrolysable radical preferably underphysiological conditions; R₂ is H, β-D-ribose, β-D-2-deoxyribose, ortheir 5′-mono-, 5′ di-, and 5′ tri-phosphate.

[0035] Suitably, the modified ligand inhibits said es nucleosidetransporter and/or nucleoside/nucleotide/nucleobase-sensitive proteinsand has a general formula selected from the group consisting of:

[0036] wherein R₄ is 4-[N-methyl]cyclohexane carboxylate,N-[m-benzoate], 4-[p-phenyl]butyrate, N-[γ-butyrate], N-[α-acetate], orN-[ε-caproylate];

[0037] wherein R₄ is 4-[N-methyl]cyclohexane carboxylate,N-[m-benzoate], 4-[p-phenyl]butyrate, N-[γ-butyrate], N-[α-acetate], orN-[ε-caproylate];

[0038] wherein R₅ is 4-carbonyl-α-methyl-α-toluene,6-[α-methyl-a-tuloamido]-hexanoate, N-[3-propionate], or6-[3′-propioamido]hexanoate; and

[0039] wherein R₅ is 4-carbonyl-α-methyl-α-toluene,6-[α-methyl-α-tuloamido]-hexanoate, N-[3-propionate], or6-[3′-propioamido]hexanoate.

[0040] In another embodiment modified ligands that bind to seratoninreceptors can be used.

[0041] In another aspect, the invention resides in a compositioncomprising the modified ligand as broadly described above, together witha pharmaceutically acceptable carrier.

[0042] In a further aspect of the invention, there is provided a methodof treatment or prophylaxis of a condition associated with a targetreceptor, said method comprising administering to a patient in need ofsuch treatment a therapeutically effective dosage of the composition asbroadly described above.

[0043] According to another aspect of the invention, there is provided amethod of detecting the presence of a target receptor in a test sample,comprising: contacting said sample with a modified ligand as broadlydescribed above, wherein said modified ligand binds said targetreceptor; and detecting the presence of a complex comprising saidmodified ligand and said receptor in said contacted sample.

[0044] In another aspect of the invention, there is provided a method ofquantifying the presence of a target receptor in a test sample,comprising: contacting said sample with a modified ligand as broadlydescribed above, wherein said modified ligand binds said targetreceptor; measuring the concentration of a complex comprising saidmodified ligand and said receptor in said contacted sample; and relatingsaid measured complex concentration to the concentration of saidreceptor in said sample.

[0045] In yet another aspect, the invention provides a method ofdetecting the presence of a target receptor on a cell or cell membrane,comprising: contacting a sample containing said cell or cell membranewith a modified ligand as broadly described above, wherein said modifiedligand binds said target receptor; and detecting the presence of acomplex comprising said modified ligand and said cell or cell membranein said contacted sample.

[0046] In another aspect of the invention, there is provided a method ofquantifying the presence of a target receptor on a cell or cellmembrane, comprising: contacting a sample containing said cell or cellmembrane with a modified ligand as broadly described above, wherein saidmodified ligand binds said target receptor; measuring the concentrationof a complex comprising said modified ligand and said cell or cellmembrane in said contacted sample; and relating said measured complexconcentration to the concentration of said receptor present on said cellor cell membrane.

[0047] In another aspect, the invention extends to a probe thatcovalently binds to a target receptor, said probe comprising a modifiedligand as broadly described above having a reporter molecule associatedtherewith.

[0048] In one embodiment, the probe comprises the modified ligand thatis interactive with es nucleoside transporter and/ornucleoside/nucleotide/nucleobase-sensitive proteins, as broadlydescribed above.

[0049] In this respect, the cell is preferably an animal cell, morepreferably a mammalian cell, and more preferably a human cell.Alternatively, the cell may be a plant cell or a microbial cell. Themicrobial cell includes, but is not restricted to, a cell of bacterial,viral or fungal origin.

[0050] The invention also encompasses the use of the modified ligand andprobe as broadly described above inter alia in the study, treatment andprevention of conditions associated with their corresponding targetreceptors.

[0051] In one embodiment, there is provided process for modifying aparent ligand, comprising attaching to said parent ligand a conjugationagent that is reactive with a moiety of a target receptor to which saidparent ligand binds, wherein when said parent ligand binds to thereceptor a covalent bond is formed between said conjugation agent andsaid moiety.

[0052] In one preferred embodiment, the conjugation agent is positionedon the ligand at a position that promotes and/or permits covalent bondformation with the moiety of the target receptor. In another preferredembodiment, the receptor to which the ligand binds is an active siteassociated with a biological activity. The receptor is, for example, acell surface receptor. The binding of the modified ligand to thereceptor in one embodiment is associated with altered activity of thetarget receptor.

[0053] In another preferred embodiment, the parent ligand and/orreceptor are naturally occurring. In another embodiment, the modifiedligand is not a crosslinking agent. In another embodiment, the modifiedligand optionally does not comprise a photo-reactive group such as aphotolabel. In another embodiment, the modified ligand does not comprisea label. In another embodiment, the receptor to which the ligand bindsis not a nucleic acid, such as RNA or DNA. In another embodiment, theconjugation agent does not bind to a moiety of a nucleic acid, andoptionally binds to a residue of an amino acid.

[0054] In one embodiment, there is provided process for modifying aparent ligand, comprising attaching to said parent ligand a conjugationagent that is reactive with a moiety of a target receptor to which saidparent ligand binds, wherein when said parent ligand binds to thereceptor a covalent bond is formed between said conjugation agent andsaid moiety, and wherein the parent ligand binds specifically with anucleoside transporter.

[0055] In another embodiment, there is provided a process for modifyinga parent ligand, comprising attaching to said parent ligand a sulfhydrylgroup specific conjugation agent that is reactive with a sulfhydrylgroup of a target receptor to which said parent ligand binds, whereinwhen said parent ligand binds to the receptor a covalent bond is formedbetween said conjugation agent and said sulfhydryl group, and whereinthe parent ligand binds specifically with a serotonin receptor.

[0056] In one embodiment, there is provided a modified ligand having thegeneral formula:

L—R₁—I—A  (I)

[0057] wherein L is a parent ligand that binds specifically with atarget receptor comprising a nucleoside transporter;

[0058] wherein A is a conjugation agent that is reactive with a moietyof the target receptor to which the parent ligand binds, such that whensaid parent ligand binds to the receptor a covalent bond is formedbetween said conjugation agent and said moiety; and R₁ is an optionalspacer.

[0059] In another embodiment, there is provided a modified ligand havingthe general formula:

L—R₁—A  (I)

[0060] wherein L is a parent ligand that binds specifically with atarget serotonin receptor;

[0061] wherein A is a conjugation agent that is reactive with asulfhydryl group of said target receptor to which the parent ligandbinds, such that when said parent ligand binds to the receptor acovalent bond is formed between said conjugation agent and saidsulfhydryl group of said receptor; and R₁ is an optional spacer.

[0062] The disclosure of all patents, patent applications, publicationsand published patent applications referred to herein are incorporatedherein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The invention, as exemplified by preferred embodiments, isdescribed with reference to the following drawings in which:

[0064]FIG. 1 shows the NEM inhibition of equilibrative ³H-uridinetransport in murine myeloma SP2/0-Ag14 cells.

[0065]FIG. 2 shows the effects of NEM on the kinetics of ³H-NBMPRequilibrium binding in murine myeloma SP2/0-Ag14 cells.

[0066]FIG. 3 shows the general reaction scheme for synthesis of CrMCC.

[0067]FIG. 4 shows the rate of CrMCC formation.

[0068]FIG. 5 shows the light absorbance profile of CrMCC and NBMPR.

[0069]FIG. 6 shows the inhibition of ³H-NBMPR binding by CrMCC, cytidineand SMCC in human HL-60 promyelocytic leukemia plasma membranes.

[0070]FIG. 7 shows the effect of CrMCC on the kinetics of ³H-NBMPRbinding to human HL-60 promyelocytic leukemia plasma membranes.

[0071]FIG. 8 shows the effects of CrMCC, cytidine and SMCC on growth ofHL-60 cells.

[0072]FIG. 9 shows the time course of ³H-CrMCC binding to human HL-60promyelocytic leukemia plasma membranes.

[0073]FIG. 10 shows the dissociation of ³H-CrMCC and ³H-cytidine fromthe binding sites of human HL-60 promyelocytic leukemia plasmamembranes.

[0074]FIG. 11 shows the concentration dependence of ³H-CrMCC binding tohuman HL-60 promyelocytic leukemia plasma membranes.

[0075]FIG. 12 shows the effect of pH on the dissociation of ³H-CrMCCfrom its binding site in human HL-60 promyelocytic leukemia plasmamembranes. FIG. 13 shows the inhibition of ³H-CrMCC binding to humanHL-60 promyelocytic leukemia plasma membranes.

[0076]FIG. 14 shows the covalent binding of sulfhydryl reactive³H-cytidine analogs to human HL-60 promyelocytic leukemia plasmamembranes.

[0077]FIG. 15 shows the UV absorbance profile of reversed-phasechromatography of human HL-60 promyelocytic leukemia plasma membraneproteins.

[0078]FIG. 16 shows the radioactivity profile of reversed-phasechromatography of human HL-60 promyelocytic leukemia plasma membraneproteins.

[0079]FIG. 17 shows the covalent binding of sulfhydryl group reactive³H-adenosine analogs to human HL-60 promyelocytic leukemia plasmamembranes.

[0080]FIG. 18 shows the inhibition of ³H-5-HT binding to murine brainmembranes by various 5-HT analogs.

[0081]FIG. 19a shows the chemical structure of LBT3001(1-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-dione)).

[0082]FIG. 19b shows the reaction scheme for synthesis of LBT3001.

[0083]FIG. 20a shows the chemical structure of LBT3002(4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-(5-hydroxyl-1H-indol-3-yl)-ethyl]-butyramide).

[0084]FIG. 20b shows the reaction scheme for synthesis of LBT3002.

[0085]FIG. 21a shows chemical structure of LBT3004(3-(2,5-dioxo-2,5-dihydro-pyrrol -1-yl)-N-[2-(5-hydroxyl-1H-indol-3-yl)-ethyl]-propionamide).

[0086]FIG. 21b shows the reaction scheme for synthesis of LBT3004.

[0087]FIG. 22a shows the chemical structure of LBT3005(4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)-cyclohexane carboxylicacid [2-(5-hydroxy-1 H-indol-3-yl)-ethyl]-amide).

[0088]FIG. 22b shows the reaction scheme for synthesis of LBT3005.

[0089]FIG. 23 shows the structures of exemplary ligands that can bemodified to include a conjugation agent.

DETAILED DESCRIPTION

[0090] 1. Definitions

[0091] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by those of ordinaryskill in the art to which the invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, preferredmethods and materials are described. For the purposes of the presentinvention, the following terms are defined below.

[0092] The articles “a” and “an” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

[0093] By “attached” is meant direct or indirect attachment of aconjugation agent to a ligand in such a manner as to resist separationof the conjugation agent from the ligand under normal physiologicalconditions. Accordingly, the term “attached” as used herein includeswithin its scope one more ionic bonds, hydrogen bonds, van der Waalsforces, covalent bonds or combinations thereof that form between theconjugation agent and the ligand or between an intervening spacer andthe conjugation agent and the ligand, respectively, such that separationof the conjugation agent from the ligand is resisted under normalphysiological conditions.

[0094] Throughout this specification, unless the context requiresotherwise, the words “comprise”, “comprises” and “comprising” will beunderstood to imply the inclusion of a stated step or element or groupof steps or elements but not the exclusion of any other step or elementor group of steps or elements.

[0095] By “conjugation agent” is meant a moiety of a modified ligandthat is reactive with a moiety of a receptor that binds a parent ligandfrom which the modified ligand was derived wherein a covalent bond isformable between the conjugation agent and the receptor moiety. In thisconnection, the conjugation agent may be sufficient either on its own orin the presence of an ancillary conjugation agent to facilitate covalentcoupling with the receptor moiety. The ancillary coupling agent may bean enzyme that catalyzes, or an activating agent that causes, formationof a covalent bond between the conjugation agent and said receptormoiety.

[0096] By “isolated” is meant material that is substantially oressentially free from components that normally accompany it in itsnative state.

[0097] As used herein, the term “ligand” refers to an agent that binds,interacts or otherwise associates with, a target receptor. The agent maybind the target receptor when the target receptor is in its nativeconformation, or when it is partially or totally unfolded or denatured.According to the present invention, a ligand is not limited to an agentthat binds a recognized functional region of the target receptor e.g.the hormone-binding site of a receptor, and the like. In practicing thepresent invention, a ligand can also be an agent that binds any surfaceor internal sequences or conformational domains of the target receptor.Preferably, the ligand is a molecule affecting physiological functionincluding a drug. The ligand can also be an endogenous ligand.

[0098] By “obtainedfrom” is meant that a sample such as, for example, anextract comprising a receptor is isolated from, or derived from, aparticular source such as a suitable cell or tissue source inclusive ofhuman, animal, plant or microbial origin.

[0099] The term “patient” as used herein refers to any organism in whichtherapy or prophylaxis of a condition associated with a receptor isdesired using the methods of the invention. However, it will beunderstood that “patient” does not imply that symptoms are present. Thepatient may therefore include a microbe, plant or animal. Preferably,the patient is a human or other mammal and includes any individual it isdesired to examine or treat using the methods of the invention. Suitablemammals that fall within the scope of the invention include, but are notrestricted to, primates, livestock animals (e.g., sheep, cows, horses,donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats,guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captivewild animals (e.g., foxes, deer, dingoes).

[0100] By “pharmaceutically acceptable carrier” is meant a solid orliquid filler, diluent or encapsulating substance that may be safelyused in topical or systemic administration.

[0101] The term “pharmaceutically acceptable salts” as used hereinrefers to non-toxic salts of the modified ligands of this invention,which are generally prepared by reacting the free base with a suitableorganic or inorganic acid. Representative salts include the followingsalts: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,bitartrate, borate, bromide, calcium edetate, camsylate, carbonate,chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynapthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,oleate, oxalate, pamaote, palmitate, panthothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, tannate, tartrate, teoclate, tosylate,triethiodide, valerate.

[0102] The term “receptor” as used herein refers to a structureincluding a molecule or a cluster of molecules that is specific for oneor more ligands wherein binding, interaction or otherwise association ofthe ligand(s) with the receptor effects, changes or nullifies a functionof the receptor. The receptor is preferably, but not exclusively, aprotein. Representative receptors include, but are not limited to, aninsulin receptor, epidermal growth factor receptors, γ-aminobutyric acidreceptors, nicotinic acetylcholine receptors, serotonin receptors, α-and β-adrenoceptors, dop amine receptors, histamine receptors,prostanoid receptors, adenosine receptors, cyclic nucleotide receptors,glutamate receptors, cytokine receptors, atrial naturetic peptidereceptors, and the prostaglandin receptors. The receptor can includetransporters such as glucose, amino acids transporters, sodium-protonexchangers, chloride-bicarbonate exchangers, sodium pumps, calciumpumps, proton pumps; channels such as sodium channels, potassiumchannels, calcium channels and chloride channels; enzymes such asoxidoreductases, transferases, hydrolases, lyases, isomerases, andligases. However, it will be understood that the receptor need not be aprotein and may include, for example, a nucleic acid in which the aminogroup found on adenine, guanine and cytosine may be targeted by aconjugation agent according to the invention. Alternatively, thereceptor may be a carbohydrate (e.g., amino-containing carbohydratessuch as aminophenyl glycosides) or a lipid (e.g., present on thephosphate head groups of some phospholipids) having one or more carboxylgroups, and/or one or more amino groups that may be targeted by aconjugation agent.

[0103] By “reporter molecule” as used in the present specification ismeant a molecule that, by its chemical nature, provides an analyticallyidentifiable signal that allows the detection of a complex comprising aligand and its cognate receptor. The term “reporter molecule” alsoextends to use of cell agglutination or inhibition of agglutination suchas red blood cells on latex beads, and the like.

[0104] The term “sample” as used herein refers to any suitable samplethat may contain a target receptor according to the invention. Thesample may be extracted, untreated, treated, diluted or concentratedfrom any suitable source and may contain one or more cells and/or cellmembranes. The sample may comprise whole cells, denatured cells,cellular membranes or parts thereof. Alternatively, the sample maycontain an isolated receptor. Suitably, the sample may comprise cellsobtained from a tissue biopsy. Alternatively, the sample may comprisecells or cell lines, which have been cultured in vitro.

[0105] The term “spacer” as used herein refers to a chemical linker,polymer, peptide and the like that spatially separates the conjugationagent from the ligand. Preferably, the spacer is selected such that itdoes not interfere with the binding of the modified ligand to thereceptor.

[0106] By “therapeutically effective amount”, in the context of thetreatment of a condition associated with a receptor, is meant theadministration of that amount to a patient in need of such treatment,either in a single dose or as part of a series, that is effective fortreatment of that condition. The effective amount will vary dependingupon the health and physical condition of the individual to be treated,the taxonomic group of individual to be treated, the formulation of thecomposition, the assessment of the medical situation, and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials.

[0107] 2. Modified Ligands

[0108] The present invention resides, at least in part, in thesurprising discovery that a conjugation agent can be attached to aparent ligand to form a modified ligand that binds irreversibly to atarget receptor to which the parent ligand binds reversibly. Theirreversible binding of the modified ligand to the receptor is effectedby formation of a covalent bond between the conjugation agent and amoiety present on the receptor, which is preferably one or morefunctional amino acid side chain groups (sometimes referred to herein as“functional groups”). The covalent bond is formed by association of themodified ligand with the receptor, followed by neighboring reactivefunctional group attack by the conjugation agent. This irreversibleinteraction of the modified ligand with the receptor results in eitherpermanent inhibition (for an antagonist) or stimulation (for an agonist)of the receptor functions. Normal function or activity of the receptorresumes only after new receptors are synthesized.

[0109] Parent ligands that can be modified include ligands that areinteractive with es nucleoside transporter and/ornucleoside/nucleotide/nucleobase-sensitive proteins. Two forms ofnucleoside transporters are classified based on their sensitivity toinhibition by NBMPR (nitrobenzylthioinosine). See Griffith et al.,Biochim. Biophys. Acta, vol. 1286, pp. 153-181 (1996). The group oftransporters sensitive to NBMPR is designated es (equilibriumsensitive), and the group insensitive is designated ei (equilibriuminsensitive). In one embodiment, compounds are provided that havedifferential binding to es vs. ei receptors. Compounds are provided thatbind selectively and irreversibly to es receptors. The expression of esreceptors is positively correlated with the carcinogenic state of cells.

[0110] Among the numerous reactive functional amino acid side chains onthe es nucleoside transporter proteins that can be used for covalentattachment, the sulfhydryl group of cysteine residue is probably themost pharmacological and biologically important. Early studies on theeffect of sulfhydryl reagents on nucleoside transport in mammalian cellshad shown to cause a marked inhibition of uridine uptake in a variety ofcultured cells (Plagemann & Richey (1974), Biochim. Biophs. Acta, vol.344, pp. 263-305; Plagemann et al. (1978), J Cell. Physiol., vol. 97,pp. 49-72; Plagemann & Wohlhueter (1980), Curr. Top. Membr. Transp.,vol. 14, pp. 225-230; Belt (1983), Biochem. Biophys. Res. Commun., vol.110, pp. 417-423; Belt (1983), Mol. Pharmacol., vol. 24, pp. 479-484).However most of the earlier studies were directed toward the totalnucleoside uptake and little attempt were made to distinguish thedifferential in sensitivity towards sulfhydryl reagents by the es and eitransport systems. Furthermore, the sulfhydryl reagents used were mainlyof organomercurial compounds, which were demonstrated to perturb plasmastructure at concentration as low as 200 μM (Belt & Noel (1985),Biochem. J., vol. 232, pp.681-688).

[0111] Ligands also may be modified which are ligands that bind toserotonin (5-hydroxytryptamine, or 5-HT) receptors. 5-HT receptorsinclude a diversity of receptor subtypes (Peroutka, S. J., CNS Drugs,(1995), vol. 4 (Suppl 1), pp. 18-28). With the exception of the 5-HT₃receptor, which is an ion channel (Derkash, V. et. al., Nature, (1989),vol. 339, pp. 706-709), other 5-HT receptors belong to the extensivefamily of seven transmembrane G protein-coupled receptors. The clinicalsignificance of the effects of 5-HT is manifest for example, inneurological, CNS, psychiatric and mood disorders, including migraine,anxiety, depression, schizophrenia, obsessive compulsive disorder,psychosis, aggression, hostility, eating disorders, gastrointestinaldisorders, hypertension, the maintenance of the circadian rhythms in thesleep-wakefulness cycle, sexual activity, compulsive behavior,temperature, emesis, and cardiovascular and motor function. Drugs thattarget the 5-HT receptors thus have wide clinical applications. Ligandsthat bind to 5-HT receptors can have effects by binding, for example, tothe cardiovascular system, platelets, gastrointestinal tract, and thebrain (Erspamer, V., Ed. “5-Hydroxytryptamine and RelatedIndolealklylamines”, Handbuch der Expermentellen Pharmakologie, Vol. 19.Springer-Verlag, Berlin, (1996), pp. 132-181).

[0112] Thus, for example, the ligand 5-HT may be modified as disclosedherein. Other ligands that can be modified include 5-HT precursors and5-HT receptor agonists and antagonists known in the art. Examplesinclude the precursor 5-hydroxytryptophan, which has been used as anantidepressant drug; 5-HT_(1A)-agonists used as tranquilizers andantihypertensives; sumatriptan, a selective 5-HT₁ receptor agonist, usedfor migraine; 5-HT₂-antagonists such as methysergide, used for migraineprophylaxis and in carcinoid tumour syndrome; 5-HT₂-antagonists such asketaserin, used to lower the blood pressure in hypertensive patients;and selective 5-HT₃-antagonists used to treat cytostatic- andradiation-induced emesis. (Beasley, CM., et. al., Psychopharmacology,(1992), vol. 107, pp. 1-10; Bolden-Watson, C., and Richelson, E., LifeSciences, (1993), vol. 52, pp. 1023-1029.; Koe, B. K., J Clin.Psychiatry, (1990), vol. 51, pp. 13-17).

[0113] Serotonin binding ligands which can be modifed include thosedeveloped for CNS disorders, such as anxiety, such as benzodiazepines,or 5-HT_(1A) agonists, such as buspirone, gepirone, ipsapirone, andflesinoxan. As anxiolytic agents, they possess advantages overbenzodiazepines because they lack the sedation and drug dependenceliabilities of bezodiazepines (Barrett, J. E., and Vanover, K. E.,Psychopharmacology, (1993), vol. 112, pp. 1-12.). Other serotoninbinding ligands include those developed for treatment of depression,such as SSRIs, for example fluoxetine. SSRIs have also been shown tohave efficacy in the treatment of bulimia and obsessive-compulsivedisorders and may be also useful in treating obesity, panic disorders,premenstrual syndrome, diabetic neuropathy, chronic pain, and certaincognitive aspects of Alzheimer's disease. Other useful antidepressantdrugs which are relatively potent antagonists at 5-HT₂ receptors,include tricyclic antidepressants, such as trazadone and nefazodone(Cowen, P. J., and Anderson. I. M, “5-Hydroxytryptamine in Psychiatry”(Sandler, M., Coppen, A., & Harnett, S. Eds.), Oxford University Press,New York, (1991)). Ligands include compounds developed for the treatmentof schizoprenia, including the antipsychotic agents clozapine,risperidone, and olanzepine, (Weil-Malherbe, H., Serotonin andschizophrenia. In “The Central Nervous System. Vol. 3, Serotonin inHealth and Disease”, Essman, W. B., Ed, Spectrum Publications, Inc., NewYork, (1978), pp. 231-291). Ligands associated with the treatment ofeating disorders can be used, such as the SSRIs and fenfluramine.Fenfluramine may be useful in the treatment of other diseases, such asautism, premenstrual syndrome, seasonal affective disorder, andattention deficit disorder. Ligands associated with alleviation ofgastrointestinal disorders can be used, such as ondansetron andgranisetron, which are used in the treatment of radiation induced emesisassociated with cancer chemotherapy, and for ameliorating the nausea andvomiting occurring during recovery from general anesthesia. Otherligands include metoclopramide and cisapride. Ligands used in control ofthe sleep-wakefulness cycle, such as L-tryptophan or nonselective 5-HTagonists or 5-HT antagonists such as ritanserin may be used (for areview, see Wauquier, A., and Dugovic, C., Ann N.Y. Acad. Sci., (1990),vol. 600, pp. 447-459). Other ligands include etanserin, a 5-HT₂receptor antagonist used as an antihypertensive agent.

[0114] 2.1. Considerations Relating to Protein Structure Reactivity

[0115] Peptides and proteins are composed of amino acids polymerizedtogether through the formation of peptide (amide) bonds. Thepeptide-bonded polymer forms the backbone of polypeptide structure.There are 20 common amino acids found throughout nature, each containingan identifying side chain of particular chemical structure, charge,hydrogen bonding capability, hydrophilicity (or hydrophobicity), andreactivity. The side chains do not participate in peptide formation andare thus free to interact and react with their environment.

[0116] Amino acids may be grouped by type depending on thecharacteristics of their side chains. The most significant amino acidsfor covalent conjugation purposes are the ones containing accessibleionizable side chains such as aspartic acid, glutamic acid, lysine,arginine, histidine, cysteine, and tyrosine. Methionine and tryptophanalso containing ionizable side chains, however, they are not easilyaccessible as they are usually buried deep inside the molecularstructure of receptors due to their hydrophobic nature.

[0117] Both aspartic and glutamic acids contain carboxylate groups thathave ionization properties. Carboxylate groups in proteins may bederivatized through the use of amide bond forming agents or throughactive ester or reactive carbonyl intermediates.

[0118] Lysine, arginine, and histidine have ionizable amine containingside chains. These amine containing side chains typically are exposed onthe surface of proteins and can be derivatized with ease. The mostimportant reactions that can occur with these residues are alkylationand acylation.

[0119] Cysteine is the only amino acid containing a sulfhydryl group.The most important reaction of cysteine groups in proteins is theformation of disulfide cross-links with another cysteine molecule.Cysteine sulfhydryls and cystine disulfides (called cystine residues)may undergo a variety of reactions, including alkylation to form stablethioether derivatives, acylation to form relatively unstable thioesters,and a number of oxidation and reduction processes. Cysteine and cystinegroups are relatively hydrophobic and usually found within the core of aprotein. It is often difficult to access the sulfhydryl groups of largeproteins without the presence of a deforming agent or a “driver”.Nevertheless, such steric hindrance does give the sulfhydryl groups aleading edge in selectivity. Thus, to access the functional reactivesulfhydryl group situated deep into the ligand-binding site, withoutusing deforming agents, is to utilize its physiological ligands or drugsto direct the conjugation agent as herein described into the innerstructure of receptors. It is possible that irreversible binding drugsthat are targeted at the sulfhydryl groups is likely to have a lowerlevel of non-specific binding, in clinical term: “fewer side effects”,than any other functional groups.

[0120] Tyrosine contains a phenolic side chain. Although the amino acidis only sparingly soluble in water, the ionizable nature of the phenolicgroup sometime makes it appear in hydrophilic regions of aprotein—usually at or near the surface. Thus unlike cysteine residue,tyrosine derivatization proceeds without much need for deforming agentsto further open the protein structure. Tyrosine may be targetedspecifically for modification through its phenolate anion by acylation.

[0121] In summary, protein molecules may contain up to nine amino acidsthat are readily derivatizable at their side chains. These nine residuescontain eight principal functional groups with sufficient reactivity formodification reactions. They are the guanidinyl group of arginine, theγ- and β-carboxyl groups of glutamic and aspartic acids, respectively,the sulfhydryl group of cysteine, the imidazolyl group of histidine, theε-amino group of lysine, the thioether moiety of methionine, the indolylgroup of typtophan and the phenolic hydroxyl group of tyrosine. Sincemethionine and tryptophan are generally buried in the interior ofproteins and are thereby protected from conjugates dissolved in thesolvent, they show only some selected reactivity in intact proteins. Theother ionizable groups are normally either exposed on the surface ofproteins or can be accessed with the help of deforming agents or“drivers”. They are therefore the easy targets for conjugation. However,among the numerous reactive functional amino acid side chains on theproteins that can be used for labeling, the sulfhydryl group of cysteineresidue is most probably both pharmacologically and biologicallyimportant. It has been reported that in most macromolecules, there is atleast one copy of reactive sulfhydryl group situated at or closed to theligand binding sites of target macromolecules. Disruption of thisreactive sulfhydryl functional group by sulfhydryl reducing agents hasbeen shown to affect the functionalities of many macromolecules.

[0122] 2.2. Moieties of Receptors Permitting Conjugation

[0123] The sulfhydryl moiety, with the thiolate ion as the activespecies, is the most reactive functional group in a protein. With apK_(a) of about 8.6, the reactivity of the thiol is expected to increasewith increasing pH, toward and above its pK_(a).

[0124] In the process of modifying a parent ligand, it is advantageousto capitalize the presence of this highly reactive sulfhydryl group,which is typically situated near or at the ligand-binding site of thereceptor Drugs or physiological ligands can be chemically modified toinclude a conjugation agent that reacts faster with the thiol group thanany other reactive functional groups. Upon association of this modifieddrug with its receptor, this sulfhydryl group directed conjugate wouldattack any sulfhydryl group that is situated within its reachableproximity; this causes covalent binding of the drug to its receptor.

[0125] In addition to sulfhydryl group, there are also other highlyreactive functional groups present on the amino acid side chains, whichcan be chemically modified. Conjugates that are reactive to thesefunctional groups will be discussed below.

[0126] 2.3. Conjugation Agents

[0127] 2.3.1. Sulfhydryl Group Specific Conjugation Agents

[0128] N-Maleimide Derivatives.

[0129] Maleimides are considered fairly specific to the sulflhydrylgroup, especially at pHs below 7 where other nucleophiles areprotonated. In acidic and near neutral solutions, the reaction rate withsimple thiols is about 100-fold faster than with the correspondingsimple amines. Although the rate increases with pH, the reaction withthe amino group also becomes significant at high pHs. The other majorcompeting reaction is the hydrolysis of maleimides to maleamic acids.However, at pH 7, the apparent rate of hydrolysis is only 3.2×10⁻⁴ min⁻¹in 0.1 M sodium phosphate buffer at 20° C., which is too slow tointerfere with the reaction with sulfhydryl groups. The rate ofdecomposition becomes significant only at pH above neutrality. Thiolundergo Michael reaction with maleimides to yield exclusively the adductto the double bond. The resulting thioether bond is very stable andcannot be cleaved under physiological conditions.

[0130] Disulfide Reagents.

[0131] Disulfide interchange occurs when sulfhydryl groups react withdisulfides. Some of the most commonly used disulfide reagents are5,5′-dithiobis-(2-nitrobenzoic acid), 4,4′-dithiodipyridine,methyl-3-nitro-2-pyridyl disulfide, and methyl-2-pyridyl disulfide. Theprotein disulfides formed are readily reverse in the presence of freemercaptan such as 2-mercaptoethanol or dithiothreitol. The reduction ofprotein disulfide into its original sulfhydryl group allows the proteinto regain its functions. Thus, irreversible binding drugs of thiscategory provide additional safety mechanism to counter varioustherapeutic complications such as over-dosing, hyper reaction, etc.

[0132] 2.3.2. Amino Group Specific Conjugation Agents

[0133] The amino group is another strong nucleophile in the protein.However, because of its abundance and omnipresence in proteins, and therelatively high pK_(a) of the ammonium ion, most of the reagents thatreact with the amino group can also react with other functionalities.Thus, it may not be an ideal target for modified ligands according tothe invention, unless the critical cysteine residues are absent from thedrug-binding site. Nevertheless, many stable acylated products are stilland only formed with the amino groups. The most common reactions ofamines are alkylation and acylation reactions. Some of the importantalkylating agents that can be incorporated into ligand structures are:α-haloacetyl compounds (e.g., haloacetate, haloacetamides), N-maleimidederivatives, aryl halides (e.g., dinitrofluorobenzene,trinitrobenzenesulfonate), aldehydes (e.g., glutaraldehyde,formaldehyde) and ketones (e.g., pyridoxal phosphate). Acylating agentsinclude, but are not restricted to, isocyanate, isothiocyanate,imidoesters, N-hydroyxlsuccinimidyl ester, ρ-nitrophenyl ester, acylchloride, and sulfonyl chloride.

[0134] 2.3.3. Carboxyl Group Specific Conjugation Agents

[0135] The most important chemical modification reactions of carboxylgroups utilize the carbodiimide-mediated process. With proteins, theoptimum pH of the reaction is about 5, which is difficult to achieve inmost physiological conditions. Other reagents such as diazoacetateesters and diazoacetamides can also be used to esterify carboxyl groups.Similar to carbodiimides, these reagents react with high specificitywith carboxyl groups of proteins under mild acid conditions.

[0136] 2.3.4. Tyrosine Specific Conjugation Agents

[0137] Tyrosine, histidine, and other aromatic residues of proteins arerich in electrons. These residues undergo electrophilic substitutionreactions at the aromatic ring. Useful electrophiles for reaction withtyrosine and histidine in proteins are diazonium compounds. Otherprotein components such as lysine, tryptophan, cysteine, and arginineresidues react very slowly, such that diazonium reagents can be regardedas tyrosine selective. Diazonium ions are generally unstable even atneutral pH and maximum reaction with the proteins is typically achievedat alkaline pH. The phenolate ion of tyrosine also reacts similar toamino groups toward acylating agents. However, the tyrosyl group isgenerally perceived as having a lower reactivity This is not because thephenolate ion has lower nucleophilicity, but because tyrosine residuesare usually buried in a protein and are, therefore, generallyinaccessible for reactions due to their hydrophobicity.

[0138] 2.3.5. Arginine Specific Conjugation Agents

[0139] A predominant reaction of the guanidinyl moiety of arginineresidues is with 1,2-dicarbonyl reagents. Commonly used vicinaldiketones include glyoxal, phenylglyoxal, 2-3-butanedione and 1,2-cyclohexanedione.

[0140] 2.3.6. Histidine Specific Conjugation Agents

[0141] While a number of alkylating agents react with the imidazolylmoiety of histidines have been referred to earlier, the rate of thesereactions is generally inferior to other nucleophiles. Even withα-haloacetate, N-carboxymethylation is generally slow in comparison withsulfhydryl groups. However, when such reactive α-halocarbonyl group isincorporated into affinity labels (e.g., p-toluenesulfonylphenylaninechloromethylketone, p-toluenesulfonyllysinechloromethylketone), specificreaction may be achieved. Beside α-haloacetyl groups, other alkylatingagents are not as reactive towards histidine. With acylating reagents,histidine forms acylated products that are generally unstable and mayundergo spontaneous hydrolysis. The most important acylating agent thathas been commonly used for the modification of histidines isdiethylpyrocarbonate or ethoxyformic anhydride. The acylated imidazoleis reversed at alkaline pH, resulting in the recovery of histidine.Deacylation can be achieved at neutral pH very rapidly withhydroxylamine.

[0142] 2.3.7. Methionine Specific Conjugation Agents

[0143] The major chemical modification reaction of methionine isalkylation. Because methionine is often situated in the hydrophobicinterior of proteins, it tends to provide high degree of selectivity.Only alkylating reagents that are coupled to the ligands are accessibleto these buried methionine residues.

[0144] 2.3.8. Tryptophan Specific Conjugation Agents

[0145] Due to its hydrophobicity, tryptophan residues are generallyburied in the interior of proteins. Tryptophan residues can be modifiedwith N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide. A distinctreagent, ρ-nitrophenylsulfenyl chloride, has been used for modificationof the indolyl moiety. The reaction is selective for tryptophan andcysteine residues.

[0146] 2.3.9. Serine Specific Conjugation Agents

[0147] Many reactive reagents such as diisopropylfluorophosphate,phenylmethyl-sulfonylfluoride and other acrylsulfonyl fluorides havebeen found to react with the active-site serine. Care should beexercised in use of such reagents because of the strong competitivereaction of hydrolysis.

[0148] 2.4. Design of Modified Ligands of the Invention

[0149] The covalent bond formation between the modified ligand and thereceptor can be catalyzed by enzymes, caused by activating agents, orfacilitated by the conjugation agent on its own. The covalent bond ispreferably formed with a functional group situated at or near theligand-binding site of the receptor. This strategy is advantageous as itensures a high degree of specificity.

[0150] The design of an irreversible binding ligand depends on thechemical, biological and molecular properties of both ligand andreceptor. In each instance, the conjugation agent to be introduced ontothe chemical structure of the ligand may be different and may require acertain configuration. In general, the ligand preferably includes afunctional group which permits attachment of a conjugation agent, orwhich is capable of modification to contain such a group, withoutaffecting the activity of the ligand to bind its receptor and to elicita biological activity. The modified ligand in this regard need not havethe same biological activity as the parent ligand (e.g., it may notrequire to be activated in vivo by some metabolic or catabolic step).

[0151] Some of the conditions and requirements to be considered forselection and configuration of the conjugation agent are as follows:

[0152] 1. Determine reaction specificity towards a particular functionalgroup of the receptor that is required for selection of the conjugationagent, e.g., amino, sulfhydryl, carboxyl, guanidinyl, imidazolyl, andother amino acid side chains. Selection will be dependent on theavailability of any functional group on the receptor to which the drugmolecule will be linked The irreversible binding conjugation agent ofthe modified ligand must be specific to that functional group.

[0153] 2. Hydrophobicity and hydrophilicity of the conjugation agent. Areceptor in a hydrophobic environment may require a hydrophobic ligandto reach the receptor. For example, a modified ligand having ahydrophilic conjugation agent may not be able to access a correspondingfunctional group situated deep inside the hydrophobic core of thereceptor.

[0154] 3. Cleavability of the conjugation agent. It may be desirable insome cases to separate a modified ligand bound to a receptor. Forexample, if a toxic drug (corresponding to a parent ligand) is to bemodified, a safety mechanism must be installed to preempt situationslike over-dosing. In this case, the use of cleavable conjugation agentswill enable the conjugation to be reversed if complication arises. Anumber of cleavable bonds may be employed for this purpose. Theseinclude disulfide bonds, amidine, mercurial group, vicinal glycol, azo,sulfone, ester and thioester linkages. In this regard, the conjugationagent itself may be cleavable or, if the conjugation agent is attachedto the modified ligand through a spacer, the spacer may be cleavable.The spacer in this regard can be selected from the group consisting of(N-succinimidyl 3-(2-pyridyldithio)propionate,succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene,3-(2-pyridyldithio)propionyl hydrazide, disuccinimidyl tartarate,N-[4-(p-azidophenylazo)-benzyol]-3-aminohexyl-N′-oxysuccinimide ester,4-4′-difluoro-3,3′-dinitrophenyl-sulfone,3-(4-azido-2-nitrobenzoylseleno)propionic acid, 2-methylmaleicanhydride.

[0155] 4. Size and geometry of the conjugation agent. Presence of theconjugation agent on a modified ligand must permit binding of thatligand to the receptor. Upon binding of the modified ligand to thereceptor, the conjugation agent must be in close proximity to thereceptor moiety so that conjugation is effected. A spacer as hereindefined may be required to bring the conjugation agent in closeproximity to the receptor moiety. In such a case, the length of thespacer will be dependent on the distance required for the conjugationagent to reach a conjugation-effective location adjacent to the receptormoiety. Structural analysis of receptor using X-ray crystallography,nuclear magnetic resonance and the like, combined with molecularmodeling, can be of assistance in identifying the receptor moiety to beconjugated and in selecting and configuring the conjugation agent on theligand chemical structure. The receptor moiety may be present in theligand-binding site of the receptor. Preferably, the receptor moiety ispresent outside the ligand-binding site of the receptor. While not beinglimited to any theory, it is possible that this latter approach may beadvantageous since it would be more likely to prevent disruption ofreceptor function upon binding with the modified ligand. On this basis,a parent agonist may be modified such that it crosslinks with itscognate receptor to thereby cause continuous stimulation of receptorfunction (“receptor turn-on”). Alternatively, a parent antagonist may bemodified such that it crosslinks with its cognate receptor to therebycause continuous inhibition of the receptor function (“receptorturn-off”)

[0156] 3. Compositions

[0157] The invention also encompasses a composition comprising themodified ligand as described herein, together with a pharmaceuticallyacceptable carrier.

[0158] The invention also features a method of treatment or prophylaxisof a condition associated with a target receptor, comprisingadministering to a patient in need of such treatment a therapeuticallyeffective dosage of the composition as broadly described above.

[0159] Depending upon the particular route of administration, a varietyof pharmaceutically acceptable carriers, well known in the art may beused. These carriers may be selected from sugars, starches, celluloseand its derivatives, malt, gelatine, talc, calcium sulphate, vegetableoils, synthetic oils, polyols, alginic acid, phosphate bufferedsolutions, emulsifiers, isotonic saline, and pyrogen-free water.

[0160] Any suitable route of administration may be employed forproviding a patient with a composition of the invention. For example,oral, rectal, parenteral, sublingual, buccal, intravenous,intra-articular, intra-muscular, intra-dermal, subcutaneous,inhalational, intraocular, intraperitoneal, intracerebroventricular,transdermal and the like may be employed.

[0161] Dosage forms include tablets, dispersions, suspensions,injections, solutions, syrups, troches, capsules, suppositories,aerosols, transdermal patches and the like. These dosage forms may alsoinclude injecting or implanting controlled releasing devices designedspecifically for this purpose or other forms of implants modified to actadditionally in this fashion. Controlled release of a modified ligandmay be effected by coating the same, for example, with hydrophobicpolymers including acrylic resins, waxes, higher aliphatic alcohols,polylactic and polyglycolic acids and certain cellulose derivatives suchas hydroxypropylmethyl cellulose. In addition, controlled release may beeffected by using other polymer matrices, liposomes and/or microspheres.

[0162] Compositions suitable for oral or parenteral administration maybe presented as discrete units such as capsules, sachets or tablets eachcontaining a pre-determined amount of one or more immunogenic agents ofthe invention, as a powder or granules or as a solution or a suspensionin an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion ora water-in-oil liquid emulsion. Such compositions may be prepared by anyof the methods of pharmacy but all methods include the step of bringinginto association one or more modified ligands as described above withthe carrier which constitutes one or more necessary ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelyadmixing the modified ligands of the invention with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product into the desired presentation.

[0163] The modified ligands may be in the form of a pharmaceuticallyacceptable salt as is known in the art.

[0164] The above compositions may be administered in a manner compatiblewith the dosage formulation, and in such amount as is therapeuticallyeffective. In this regard, the dose of modified ligand administered to apatient should be sufficient to effect a beneficial response in thepatient over time such as an amelioration of the condition to betreated. The quantity of the modified ligand(s) to be administered maydepend on the subject to be treated inclusive of the age, sex, weightand general health condition thereof. In this regard, precise amounts ofthe modified ligand(s) for administration will depend on the judgementof the practitioner. In determining the effective amount of the modifiedligand to be administered in the treatment or prophylaxis of thecondition associated with the target receptor, the physician mayevaluate progression of the condition.

[0165] In any event, those of skill in the art may readily determinesuitable dosages of the modified ligands of the invention. Such dosagesmay be in the order of nanograms to milligrams of the modified ligandsof the invention.

[0166] 4. Detection of Target Receptors and Cells or Cell MembranesContaining Same

[0167] The invention also features a method of detecting the presence ofa target receptor in a test sample. The method comprises contacting thesample with a modified ligand as described in Section 2, wherein saidmodified ligand binds the target receptor, and detecting the presence ofa complex comprising said modified ligand and said receptor in thecontacted sample.

[0168] The invention also encompasses a method of quantifying thepresence of a target receptor in a test sample. The method comprisescontacting the sample with a modified ligand as broadly described above,wherein the modified ligand binds said target receptor, measuring theconcentration of a complex comprising the modified ligand and thereceptor in the contacted sample, and relating the measured complexconcentration to the concentration of the receptor in the sample.

[0169] The invention also provides a method of detecting the presence ofa target receptor on a cell or cell membrane. The method comprisescontacting a sample containing the cell or cell membrane with a modifiedligand as broadly described above, wherein the modified ligand binds thetarget receptor, and detecting the presence of a complex comprising themodified ligand and the cell or cell membrane in the contacted sample.

[0170] Also encompassed by the invention is a method of quantifying thepresence of a target receptor on a cell or cell membrane. The methodcomprises contacting a sample containing the cell or cell membrane witha modified ligand as broadly described above, wherein the modifiedligand binds said target receptor. The concentration of a complexcomprising the modified ligand and the cell or cell membrane is thenmeasured in the contacted sample, and the measured complex concentrationis related to the concentration of the receptor present on the cell orcell membrane.

[0171] The modified ligands can be used as a tool to identify the actualdrug pocket or drug binding area on the receptor molecules. Since thebinding of these modified ligands to their targets is irreversible, theactual site that the drugs interact with can be identified on thereceptors using various techniques such as peptide finger printing usingMass Spec. This information can be used to identify molecules that bindto that particular drug pocket.

[0172] Any suitable technique for determining formation of the complexmay be used. For example, a modified ligand according to the invention,having a reporter molecule associated therewith (sometimes referred toherein as a “probe”) may be utilised in any suitable assay known in theart for detecting and/or quantifying ligand-receptor interactions. Forexample, scintillation counting, autoradiography, fluorography, flowcytometry, UV spectroscopy, fluorescence spectroscopy, chemiluminescenceimaging, fluorescence microscopy, confocal microscopy, electronmicroscopy, etc may be used in this regard.

[0173] The reporter molecule may be associated with the any suitablepart of the modified ligand including the conjugation agent and thespacer, if included. Preferably association of the reporter moleculewith the modified ligand is selected such that the reporter moleculedoes not interfere with binding of the modified ligand to the receptor.

[0174] It will be appreciated that the reporter molecule associated withthe antigen-binding molecule may include the following:

[0175] direct attachment of the reporter molecule to the modifiedligand;

[0176] indirect attachment of the reporter molecule to the modifiedligand; i.e., attachment of the reporter molecule to another assayreagent which subsequently binds to the modified ligand; and

[0177] attachment to a subsequent reaction product of the modifiedligand.

[0178] The reporter molecule may be selected from a group including achromogen, a chromophore, a catalyst, an enzyme, a fluorochrome, achemiluminescent molecule, a lanthanide ion such as Europium (Eu³⁴), aradioisotope, a spin label, and a direct visual label.

[0179] In the case of a direct visual label, use may be made of acolloidal metallic or non-metallic particle, a dye particle, an enzymeor a substrate, an organic polymer, a latex particle, a liposome, orother vesicle containing a signal producing substance and the like.

[0180] A large number of enzymes suitable for use as reporter moleculesis disclosed in U.S. Pat. No. 4,366,241, U.S. 4,843,000, and U.S.4,849,338. Suitable enzymes useful in the present invention includealkaline phosphatase, horseradish peroxidase, luciferase,β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and thelike. The enzymes may be used alone or in combination with a secondenzyme that is in solution.

[0181] Suitable fluorochromes include, but are not limited to,fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate(TRITC), R-Phycoerythrin (RPE), and Texas Red. Other exemplaryfluorochromes include those discussed by Dower et al. (InternationalPublication WO 93/06121). Reference also may be made to thefluorochromes described in U.S. Pat. No. 5,573,909 (Singer et al), U.S.Pat. No. 5,326,692 (Brinkley et al). Alternatively, reference may bemade to the fluorochromes described in U.S. Pat. Nos. 5,227,487,5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517,5,459,276, 5,516,864, 5,648,270 and 5,723,218.

[0182] Preferably, the reporter molecule is a radioisotope such as ³H,125I, ¹⁴C, ³²P, ³³P, and ³⁵S.

[0183] 5. Applications

[0184] Therapeutic.

[0185] The modified ligands of the invention, which preferably representirreversible binding drugs, have substantial advantages overconventional reversible binding drugs. Firstly, the dose-relatedinhibition or stimulation can be persisted long after the drugsdisappear from the plasma. In other words, the drug effect is likely tolast longer than would be predicted from its plasma eliminationhalf-life. Secondly, due to longer lasting drug effects, theseirreversible binding drugs can be administered less frequently and at alower dosage. This minimizes adverse side effect and prevent cumulativetoxicity induce by the drug themselves or by their metabolites. Thirdly,since the binding of the irreversible antagonist to its receptor ispermanent, the blockade of receptor response is not longer a competitiveinhibition mechanism. This irreversible antagonism prevents the agonist,at any concentration, from producing a maximum effect on a givenreceptor. Furthermore, if the modified ligand is rendered highlyradioactive, it may be used as a therapeutic for specifically killingcells bearing the cognate receptor or may be used for imaging.

[0186] Diagnostic.

[0187] Another application of this ligand modification technology is tofurther elucidate various receptor subpopulations that can be targetedto relieve dysfunctions in the various complex physiological processesMore importantly, the modified drugs allow us to develop/identify animalmodels that are “deficient” in certain receptors without undergoinglengthy, tedious and complicated manipulation of the genetic materialsHence the complex physiological mechanisms and functions of variousreceptors in “real-life” situations can be studied and analyzed.Furthermore, one can also study how the various different receptors areinterlinked and influenced by each other's functions. The availabilityof such animal models will also enable investigators to predict andreveal therapeutic outcomes of various drugs by simultaneously blockingmultiple receptor populations of interest. Thus, the present inventioncan be used to profile different receptors present on a cell as well asin a tissue, organ or system. Such receptor profiling can be usedadvantageously to discover novel drug targets, to predict the possibleside-effects of drugs and to determine how various cells communicatewith each other, their state of health, and whether they respond tocertain external stimuli (e.g., to drugs).

[0188] The invention will now be described with reference to thefollowing non-limiting examples.

EXAMPLES Example 1:

[0189] Effects of N-ethylmaleimide on the Equilibrative NucleosideTransporter in Murine Myeloma SP2/0-Ag14 Cells.

[0190] Exemplary N-maleimide derivatives were used as a sulfhydrylreagent that is advantageously specific to the sulfhydryl group,reacting only with certain accessible sulfhydryl groups on the proteins,making it possible for specific inhibitions, and good penetration intocells due to the uncharged nature of the compound. N-Ethylmaleimide(NEM) is the smallest maleimide reagent capable of forming stable thioesters with the reactive sulfhydryl groups of proteins. The sensitivityof es and ei nucleoside transport systems towards NEM was demonstrated.Various different cell lines were used to show that the sensitivity ofnucleoside transport system(s) toward NEM is a general phenomenon andnot restricted to certain cell or tissues types.

[0191] Previous studies of the es nucleoside transporter system(Paterson et al. (1977), Mol. Pharmacol., vol. 13, pp. 114; Gati et al.(1983), Mol. Pharmacol., vol. 23, pp. 146-1520) suggested that the sugarcomponent of nucleosides was important for binding of a nucleoside tothe es nucleoside transporter site. Thus in the present invention, thatlinker moiety was attached to the pyrimidine/purine ring of thenucleosides to avoid destruction of effective inhibition for the esnucleoside transporter to provide novel probes for that transporterregulatory site.

[0192] The present inventor has found that a novel group of nucleosides,cytidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylic acid (CrMCC) andderivatives and analogues thereof, have the ability to irreversibleinhibiting the es nucleoside transporter proteins of human cells. Adirect, rapid synthetic route used to synthesize CrMCC or1-[[4-[(4-amino-1-β-D-ribofuranosyl-2(1H)-pyrimidione)carbonyl]cyclohexyl]methyl]-1H-pyrrole-2,5-dione, is set out in FIG. 3.

[0193] To demonstrate the sensitivity of es and ei nucleoside transportsystems to NEM, various mammalian cell lines were treated with varyingconcentrations of NEM from 0 to 30 mM for 1 min prior transport assay.FIG. 1 shows the equilibrative transport (measured at 5 s uptakeinterval) of ³H-uridine (final concentration 50 μM) in murine myelomaSP2/0-Ag14 cells was inhibited by NEM in an apparent biphasic manner.About 60-70% of the transport activity was inhibited by NEM with IC₅₀value of 0.15 mM. The remaining 30-40% of transport activity wasgradually abolished by NEM at concentrations above 3 mM. Similarbiphasic inhibition by NEM was also observed in human HL-60 and humanMCF-7 cells, which possess both the es and ei transport systems (Lee etaL (1995), Biochim. Biophys. Acta, vol. 1268, pp. 200-208). For cellsthat possess only es (murine EL-4 cells) or only ei (rat Morris 7777)transport system, monophasic ³H-uridine transport curves in NEMdose-response experiments were observed. The IC₅₀ values wereapproximately 0.1 and 1.5 mM for EL-4 and Morris 7777 cells,respectively. These results suggested that the biphasic curve of total³H-uridine transport observed in NEM dose-response experiments is areflection of the presence of two distinct equilibrative nucleosidetransport systems in those cells. The NEM-sensitive component is the estransport system and the NEM-insensitive component is the ei transportsystem. The ei transporter, although less sensitive to NEM inhibition,can be inhibited at higher concentrations of NEM or by prolongedexposure to NEM. This observation is in agreement with the generalnotion that sulfhydryl groups of enzymes display a considerablevariation in their reactivity, ranging from unreactive, through severalstages of sluggishness, to free and being immediately reactive.

[0194] To further demonstrate that the reduction in uridine influx by estransport system is due to NEM-induced chemical modification on thecarrier protein which subsequently affected the transport affinity, andto confirm the notion that the changes in es nucleoside transportactivity can be demonstrated by changes in ³H-NBMPR binding ability,murine myeloma SP2/0-Ag14 cells pretreated with or without 0.3 mM NEMfor 1 min were assayed for the availability of high-affinity ³H-NBMPRbinding sites on the cell membranes. FIG. 2 shows that the K_(d) value(corrected for non-specific binding determined in the presence of 20 μMof NBTGR) for ³H-NBMPR binding was changed significantly in response toNEM treatment. Shortly after 1 min of NEM exposure, the apparent K_(d)value was 1.9±0.4 nM, as compared to 0.16±0.02 nM for untreated cells.However, the Bmax values of 40,000±2,600 and 41,000±1,000 sites/cell forNEM treated and untreated cells, respectively, were not significantlydifferent. These results provide additional evidence that a criticaldisulfhydryl bond is located near or close to the nucleosidetransporting/binding site of the es transport system, where formation ofdisulfhydryl bond with NEM affects the transport affinity of thecarrier. The suggestion that the critical sulfhydryl group on the estransporter protein is probably located or close to but not on thenucleoside transporting/binding site is derived from the observationsthat NBMPR, dilazep, dipyridamole at 30 μM and uridine at 10 mM wereincapable of protecting this sulffiydryl group from NEM modification(Lee et al. (1995), Biochim. Biophys. Acta, vol. 1268, pp. 200-208).Although NEM is effective as an inhibitor of es transporter protein, itis toxic and should be modified for therapeutic purposes.

Example 2

[0195] Synthesis and Characterization of CrMCC

[0196] The strategy to selectively irreversibly inhibit the estransporter protein is to attach a reactive group specific covalentbinding agent (maleimide) to a driver so that it can deliver thecovalent binding agent to the desired target. The most suitable driverwill be the physiological ligand itself (nucleoside). Cytidine, apyrimidine nucleoside, is selected among other physiological nucleosidesdue to its “function inertness”, thus minimizes “non-specific” drugbinding. As for the spacer arm that links the maleimide to the cytidine,a cyclohexane carboxylic acid is chosen for pilot studies. Thisconfiguration is to mimic the chemical structure of NBMPR (FIG. 5b).Furthermore, other advantages like hydrophobicity provided bycyclohexane) and stability (provided by carboxylic acid) are also takinginto consideration.

[0197] A direct, rapid synthetic route used to synthesize CrMCC or1-[[4-[(4-amino-1-β-D-ribofuranosyl-2(1H)-pyrimidione)carbonyl]cyclohexyl]methyl]-1H-pyrrole-2,5-dione,is set out in FIG. 3.

[0198] N-Succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate(SMCC) and cytidine were dissolved in anhydrous dimethysulfoxide (DMSO)separately prior reaction. The reaction started when these two reagentswere mixed at room temperature and shielded from light. The molarconcentration ratio of SMCC:cytidine in the mixture was 0.10 M:0.15 Mwith pH 7.5-8.0 in the reaction system. Within 4 hrs, CrMCC was found inthe reaction mixture and can be separated from cytidine and SMCC by aC₁₈ reversed-phase column (Resource RPC, Pharmacia) operated on a HPLC(AKTA Purifier, Pharmacia) using the absorbance wavelength of 300 nm.Cytidine was eluted by 100% water and CrMCC and SMCC were eluted by 15%acetonitrile in water with the flow rate 1.5 ml/min. The hydrophobicityof CrMCC is greater than that of cytidine but is less than that of SMCC.Formation of CrMCC reached its near maximum after 48 hrs of reactionbetween SMCC and cytidine at 22-24° C. (FIG. 4). The area under the peakwas calculated using a computer program UNICORN (version 2.0).

[0199] HPLC purified CrMCC was lyophilized by freeze-drying. Theactivity of CrMCC was stable for at least three months if it was storedat −20° C. in a desiccator. The molecular weight of CrMCC was determinedby LC-MS (Waters). Briefly, CrMCC was eluted out of a C₁₈ reversed-phasecolumn (Resource RPC, Pharmacia) by 20% acetonitrile in water at aflow-rate of 3 ml/min. The capillary voltage of micromass massspectrometer was set to 3.0-3.5 V, and the cone voltage was set at 20 V.N₂ gas flow was at 700 L/hr and electrospray was negative. Mass spectrawere gathered under a full-scan operation, scanning range 1-1000 m/z.The molecular weight of CrMCC was determined by monitoring theprotonated molecular ion, and was similar to the predicted value of462.5. The purity of CrMCC synthesized was consistently greater than95%.

[0200] The light absorbance spectrum of CrMCC was measured by uv-visspectrophotometer. FIG. 5A shows there are two λ_(max)'s for the lightabsorbance spectrum of CrMCC. One of them is at 254 nm (similar to theλ_(max) of cytidine) and the other one is at 300 nm (similar to theλ_(max) of SMCC). Such an absorbance profile of CrMCC with two λ_(max)'sis very similar to that of NBMPR (FIG. 5B). This indicated that CrMCCconsists of both pyrimidine and cyclohexane rings on its chemicalstructure. Subsequent structural studies using NMR confirmed thisfinding.

Example 3

[0201] Effects of CrMCC on the Binding of ³H-NBMPR in Human HL-60Promyelocytic Leukemia Plasma Membranes

[0202] To synthesize ³H-CrMCC, radioactive cytidine (³H-cytidine) wasused. Since high concentration of substrate increases the yield ofCrMCC, nonradioactive cytidine was pre-mixed with radioactive³H-cytidine at a concentration ratio of 100:1 prior to reaction withSMCC (see Example 2).

[0203] Purified HL-60 plasma membranes suspended in reaction buffer(0.13 M NaCl, 0.02 M NaHCO₃, pH 7.0) were pretreated with gradedconcentrations of cytidine, SMCC, and CrMCC for 5 min prior exposed to³H-NBMPR (5 μM) for additional 30 min. The reaction was terminated bymembrane filtration method (Lee & Jarvis (1988), Biochem. J., vol 249,pp. 557-564). The data shown in FIG. 6 were corrected for non-specificbinding determined in the presence of 20 μM of NBTGR. FIG. 6 shows thespecific of ³H-NBMPR to HL-60 unsealed plasma membranes was inhibited byCrMCC with IC₅₀ value of 10 μM. In contrast, the IC₅₀ values for SMCCand cytidine in inhibiting ³H-NBMPR binding were 100 μM and >1 mM,respectively. The Ki value for CrMCC was calculated to be 1 μM.

[0204] To analyse the effect of CrMCC on the binding kinetics of³H-NBMPR, purified HL-60 plasma membranes were pretreated with 0, 10,and 50 μM of CrMCC for 5 min prior incubated with graded concentrationsof ³H-NBMPR (0.2 to 8 nM) for additional 30 min. A double reciprocalplot of the results is presented in FIG. 7. The lines of the plots wereintersected at the abscissa indicating a changed value for B_(max) butan unchanged value for K_(d) in the presence of CrMCC. For the datashown, the apparent K_(d) values of ³H-NBMPR binding were 0.36±0.04,0.31±0.03 and 0.39±0.05 nM with B_(max) values of 1.56±0.05, 0.59±0.01and 0.30±0.01 pmol/mg protein for membranes treated with 0, 10 and 50 μMof CrMCC, respectively. Data were corrected for non-specific bindingdetermined in the presence of 20 μM of nitrobenzylthioguanosine (NBTGR),a non-radioactive competitive ligand. These results suggested anon-competitive inhibition of ³H-NBMPR binding by CrMCC. This is aunique feature of the irreversible antagonism.

[0205] Any clinically useful drugs must have little or no cytotoxicityat their therapeutic dosage range. HL-60 cells in logarithmic growth atan initial cell density of 5×10⁴ cells/ml in RPMI medium containing 10%FBS were exposed to graded concentrations of CrMCC, cytidine, and SMCC(0 to 100 μM) for 3 days. The cell density was counted using anelectronic particle analyzer (Sysmex). FIG. 8 shows both CrMCC andcytidine had little or no effect on HL-60 cell growth at concentrationsas high as 100 μM after 3 days of exposure. In contrast, SMCC, one ofthe parent compounds, was extremely toxic to HL-60 cells with IC₅₀ valueof less than 0.5 μM on cell growth. The toxicity of SMCC is attributedto its non-specific interaction with every accessible sulfhydryl groupson the cells. Little or no inhibition on cell growth by cytidine isexpected as this nucleoside is rather “inert” and does not inducenucleotide imbalance at concentration range tested

Example 4

[0206] Binding of ³H-CrMCC to the Human HL-60 Promyelocytic LeukemiaPlasma Membranes

[0207] The availability of radioactive CrMCC (³H-CrMCC) makes itpossible to study the biochemical properties of CrMCC on the esnucleoside transporter. This experiment was conducted to investigate therate of ³H-CrMCC binding to the unsealed plasma membranes of HL-60cells. FIG. 9 shows the binding of ³H-CrMCC (30 μM final concentration)to purified HL-60 plasma membranes was rather slowed. A minimum of 5 minis needed to achieve the maximum binding value of 12 nmoles per mg ofHL-60 plasma membrane protein. This is unlike the binding of reversibleantagonists such as NBMPR, dipyridamole and dilazep, which the bindingwas known to be rapid and mostly completed within first minute ofincubation. The binding reaction in FIG. 9 was terminated by membranefiltration method and the data were corrected for filter blanks.

[0208] It is important to confirm the binding of ³H-CrMCC to theunsealed HL-60 plasma membranes is indeed irreversible. Purifiedunsealed HL-60 plasma membranes were incubated with 30 μM of ³H-CrMCCfor 10 min. After which the mixtures were diluted 20 folds and thediluted mixtures were sat at room temperature for various time intervalsto allow dissociation to occur. FIG. 10 shows little or no dissociationof ³H-CrMCC from its binding sites occurred for at least 60 min after a20-fold dilution. Even 1 mM of cytidine presence in the dilution mediumfailed to displace ³H-CrMCC from its binding sites. In contrast, thebinding of 30 μM of ³H-cytidine to HL-60 plasma membranes was low anddissociated rapidly and completely upon dilution (inset of FIG. 10).This finding together with the non-competitive inhibition of ³H-NBMPRbinding by CrMCC (FIG. 7) suggested the interaction of CrMCC to itsbinding sites is indeed irreversible. The dissociation reaction shown inFIG. 10 was terminated by membrane filtration method and the data werecorrected for filter blanks. To determine the concentration dependenceof ³H-CrMCC binding to its binding sites, purified unsealed HL-60 plasmamembranes were incubated with graded concentration of ³H-CrMCC (3 to 800μM) for 10 min and the reaction was terminated by membrane filtrationmethod (FIG. 11). The data can be resolved into at least two components,a high affinity component (K_(d)=23.8±2.2 μM) and a low affinitycomponent (inset of FIG. 11). The kinetic constants for the low affinitycomponent cannot be estimated with the existing concentration range of³H-CrMCC. It is also possible that this low affinity component is anon-specific ³H-CrMCC binding site. The data shown in FIG. 11 werecorrected for filter blanks.

[0209] It is important to determine the stability of ³H-CrMCC-receptorcomplexes in various pH conditions. Purified unsealed HL-60 plasmamembranes were incubated with 30 μM of ³H-CrMCC for 5 min. The reactionmixture was then diluted 20-fold with reaction buffer of various pHvalues (pH 0.85 to 7.5). The dissociation reaction was terminated bymembrane filtration method. The data were corrected for filter blanks.FIG. 12 shows ³H-CrMCC-receptor complexes were very stable atphysiological pH. However, the ligand-receptor complexes began to breakdown at pH below 3.5.

[0210] Early studies with sulfhydryl reagent NEM had suggested that acysteine residue is probably situated very close to but not on thenucleoside transporting/binding site of the es nucleoside transporterprotein. Thus, if ³H-CrMCC is bind to the same site that NEM binds, thenthe substrates that failed to inhibit NEM action (Lee et al. (1995),Biochim. Biophys. Acta, vol. 1268, pp. 200-208) should similarly havelittle or no effect on ³H-CrMCC binding. FIG. 13 shows 1 mM ofphysiological nucleosides (e.g. cytidine, uridine, adenosine andinosine), and 20 μM of es nucleoside transport inhibitors (e.g. NBMPRand dipyridamole) indeed failed to inhibit the binding of ³H-CrMCC (30μM final concentration) to the HL-60 plasma membranes. In contrast, 1 mMof NEM was as effective as 0.5 mM of CrMCC in inhibiting the binding of³H-CrMCC. This finding suggested that NEM and CrMCC reacted to the samesulfhydryl group on the es nucleoside transporter protein.

Example 5

[0211] Effects of other Sulfhydrl Reactive Covalent Arms on the Bindingof ³H-cytidine Molecule to Human HL-60 Promyelocytic Leukemia PlasmaMembranes

[0212] It was successfully demonstrated that maleimide, a sulfhydrylgroup reactive agent, linked to cytidine via a cyclohexane carboxylicacid spacer arm is effective in irreversibly inhibiting the binding of³H-NBMPR to the es transporter protein. In this experiment, the bindingcapability of ³H-cytidine molecule to the HL-60 plasma membranes afterbeing linked to the same or different sulfhydryl group reactive agentvia different spacer arms was compared, to provide an indirectindication on the suitability of the spacer arms used in creating theirreversible es nucleoside transport inhibitor. Thus, ³H-cytidine waschemically modified to incorporate various sulfhydryl reactive side armsonto the 6-amino position of the pyrimidine ring. Chemicals used tomodify ³H-cytidine include m-maleimidobenzoyl-N-hydroxysuccinimide(MBS), N-succinimidyl 4-[p-maleimidophenyl]butyrate (SMPB),N-[γ-maleimidobutyryloxy]succinimide,4-suceinimidyloxycarbonyl-α-methyl-α-[2-pyridylthio]toluene (SMPT),N-succinimidyl 3-[2-pyridyldithio] propionate (SPDP), N-succinimidylmaleimidoacetate (AMAS), and N-[ε-maleimidocaproyloxy]succinimide (EMCS), in addition to N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Purified HL-60plasma membranes were incubated with 100 μM of these chemically modified³H-cytidine analogs for 5 min. The reaction was terminated by membranefiltration method. FIG. 14 shows N-α-maleimidoacetoxylic acid (AMA)covalent arm promotes highest irreversible binding of ³H-cytidine to theHL-60 plasma membranes followed by 3-[2-pyridyldithio]propionic acid(PDP) However, N-ε-maleimidocaproylic acid (EMC), m-maleimidobenzoicacid (MB), N γ-maleimidobutyrylic acid (GMB) and4-[p-maleimidophenyl]butyrylic acid (MPB) were equally effective ascovalent arms as compare to N-maleimidomethyl cyclohexane carboxylicacid (MCC). In contrast, α-methyl-α-[2-pyridyldithio]toluene carbonicacid (MPT) was least effective. Symbol X in FIG. 14 represents cytidine.

Example 6

[0213] Identification of ³H-CrMCC Binding Proteins in Human HL-60Promyelocytic Leukemia Plasma Membranes

[0214] Attempts were made to identify the plasma membrane proteins thatwere labelled by ³H-CrMCC. ³H-CrMCC (20 μM) labelled HL-60 plasmamembrane proteins solubilized in SDS were loaded onto a C₁₈reversed-phase column (Resource RPC, Pharmacia) operated on a FPLC (ATKAFPLC, Pharmacia) with the following conditions: column volume (CV, 3ml), starting buffer A (0.05% TFA in water), eluent B (0.065% TFA inacetonitrile), flow rate (2 ml/min), detection (280 nm), elution (0% Bin 3 CV, 0-5% B in 1 CV, 5% B in 5 CV, 5-100% B in 15 CV, wash-out 100%B). FIG. 15 shows the general UV (λ=280 nm) absorbance profile of HL-60plasma membranes after being separated by FPLC using a C₁₈reversed-phase column. The arrows on the chromatogram indicate theprotein peaks that were labelled by ³H-CrMCC (refer to FIG. 16). Theeluents were collected at 1 mmin and the radioactivity was determined bya liquid scintillation counter. FIG. 16 shows there are at least threemajor radioactive peaks located at fraction numbers 43-44, 49-50, and57-59 of the chromatogram. The sharp radioactive peak at fraction number6 is due to degradation product of ³H-CrMCC (i.e. ³H-cytidine) and thenon-specific binding of ³H-CrMCC to the membrane lipids.

Example 7

[0215] Development of Irreversible Binding Drugs of Adenosine Origin

[0216] In view of the success of creating irreversible inhibitor of estransporter protein using cytidine as a lead compound, reproduction ofthe invention can be implemented by attaching a covalent arm to otherphysiological nucleosides such as adenosine (FIG. 17). ³H-Adenosine waschemically modified to incorporate various sulfhydryl reactive covalentarms onto the 6-amino position of the purine ring according to thegeneral procedure set out in FIG. 3, and using the chemicals listed inExample 5. HL-60 plasma membranes were incubated with 100 μM of thesechemically modified ³H-adenosine analogs for 5 min. The reaction wasterminated by membrane filtration method. FIG. 17 shows4-[p-maleimidophenyl]butyrylic acid (MPB) covalent arm promotes highestirreversible binding of ³H-adenosine (100 μM) to the HL-60 plasmamembranes followed by N-α-maleimidoacetic acid (AMA). However,3-[2-pyridyldithio]-propionic acid (PDP), N-ε-maleimidocaproylic acid(EMC), m-maleimidobenzoic acid (MB), and N-maleimidomethyl cyclohexanecarboxylic acid (MCC) were equally lesser effective as covalent arms. Incontrast, N-γ-maleimidobutyrylic acid (GMB) andα-methyl-α-[2-pyridyldithio]toluene carbonic acid (MPT) were leasteffective.

Example 8

[0217] Inhibitory Effects of Irreversible Interaction 5-HT Analogs onthe Binding of ³H-5-HT in Murine Brain Membranes

[0218] A. Compound Synthesis

[0219] The compounds shown in FIGS. 19a, 20 a, 21 a and 22 a (chemicalstructures of LBT3001(1-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-dione), LBT3002(4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-(5-hydroxyl-lH-indol-3-yl)-ethyl]-butyramide),LBT3004(3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-(5-hydroxyl-1H-indol-3-yl)-ethyl]-propionamide),and LBT3005 (4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)-cyclohexanecarboxylic acid [2-(5-hydroxy-1H-indol-3-yl)-ethyl]-amide),respectively) demonstrated efficacy as irreversible binding compounds,and provide examples of modified ligands which are modified 5-HTreceptor binding ligands comprising conjugation agents.

[0220] The reaction scheme for synthesis of LBT3001 (FIG. 19b) is asfollows: 5-Hydroxytryptamine (212.7 mg, 1.0 mmol) was dissolved insaturated sodium bicarbonate (25 ml) solution at 0° C.N-Ethoxycarbonyleimide (177.6 mg, 1.05 mmol) was added under stirring.After 30 minutes, the ice-water bath was replaced with warm water bath(30° C. to 40° C.). The reaction solution was then stirred in the warmwater bath for about one hour. The solution was then extracted withethyl acetate (3×50 ml). The ethyl acetate layer was then washed withdeionized water until pH close to neutral (2×20 ml) and then with brine(20 ml). The organic layer was then dried with anhydrous MgSO₄. Thesolvent was removed under reduced pressure to obtain a crude product inyellow oil. Purification through flash chromatography (30% ethyl acetatein hexane) afforded the product as a yellow to orange crystal (187.2 mg,73%).

[0221] The reaction scheme for synthesis of LBT3002 (FIG. 20b) is asfollows: 5-Hydroxytryptamine hydrochloride (42.5 mg, 0.2 mmol) and3-maleimidobutyric acid (40.3 mg, 0.22 mmol) were suspended in 1 ml2-methoxyethyl ether (DME). To the solution, N-methyl morpholine (NMM,25 μl, 0.22 mmol) and 1,3-dicyclohexylcarbodiimide (DCC, 45.4 mg, 0.22mmol) were added. The solution was stirred for 3 hours at the roomtemperature. The product was purified by flash chromatography directlyusing a gradient solvent eluent (0% to 3% methanol in methylenechloride) to generate brownish oil (40.7 mg, 60%).

[0222] The reaction scheme for synthesis of LBT3004 (FIG. 21b) is asfollow: 5-Hydroxytryptamine hydrochloride (42.5 mg, 0.2 mmol) and3-maleimidopropionic acid (37.2 mg, 0.22 mmol) were suspended in 1 mlmethoxyethyl ether (DME). To the solution, N-methyl morpholine (NMM, 25μl, 0.22 mmol) and 1,3-dicyclohexylcarbodiimide (DCC, 45.4 mg, 0.22mmol) were added. The solution was stirred for 3 hours at the roomtemperature. The product was purified by flash chromatography directlyusing a gradient solvent eluent (0% to 1.5% to 5% methanol in methylenechloride) to generate an orange crystal (46.0 mg, 70%). The reactionscheme for synthesis of LBT3005 (FIG. 22b) is as follow:4-Aminomethyl-cyclohexanecarboxylic acid (2.83 g, 18.0 mmol) insaturated NaHCO₃ (80 ml) was stirred vigorously at 0° C. To thesolution, N-ethoxycarbonyleimide (finely grounded, 3.05 g, 18.0 mmol)was added portion by portion. When the addition was completed (c.a. 10minutes), deionized water (80 ml) was added and the mixture was stirredat room temperature for 40 minutes. The resulting mixture was bought topH 1-2 with 1 M of HCl and extracted with ethyl acetate (3×80 ml). Theorganic layer was washed with deionized water (50 ml) and then brine (20ml). The organic phase was dried over magnesium sulfate and evaporatedunder reduced pressure to generate a crude product. Purification byflash chromatography (hexane: ethyl acetate : acetic acid, 60: 39: 1) toafford the product as a solid (2.69 g, 63%). 5-Hydroxytryptaminehydrochloride (42.5 mg, 0.2 mmol) and maleimidomethyl-cyclohexanecarboxylic acid (52.2 mg, 0.22 mmol) were suspended in 1 ml methoxyethylether (DME). To the solution, N-methyl morpholine (NMM, 25 μl, 0.22mmol) and 1,3-dicyclohexylcarbodiimide (DCC, 45.4 mg, 0.22 mmol) wereadded. The solution was stirred for 3 hours at the room temperature. Theproduct was purified by flash chromatography directly using a gradientsolvent eluent (ethyl acetate: hexane (1:1) to ethyl acetate: hexane(3:1)) to generate a light brown oil (28.3 mg, 35.8%).

[0223] Other 5-HT receptor binding compounds such as those shown in FIG.23 can be similarly modified for irreversible binding to their targetsites using one of the reaction schemes illustrated in FIGS. 19b to 22b.

[0224] B. Assay

[0225]FIG. 18 shows the inhibitory effects of various 5-HT analogs onthe high affinity binding of ³H-5-HT to murine brain membranes. Purifiedmurine brain membranes suspended in reaction buffer (0.13 M NaCl, 0.02 MNaHCO₃, pH 7.0) were pretreated with graded concentrations of LBT3001(▪), LBT3002 (□), LBT3004 (∘), and LBT3005 () for 5 min prior exposureto ³H-5-HT (5 nM final concentration) for additional 30 min. Thereaction was terminated by membrane vacuum filtration method. The datashown were corrected for non-specific binding determined in the presenceof 1 mM of non-radioactive 5-HT. The results were plotted againstcontrol binding determined in the absence of inhibitors.

[0226]FIG. 18 shows the binding of ³H-5-HT to murine brain membranes wasinhibited by all analogs of 5-HT in an apparent biphasic manner.

[0227] LBT3001(1-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-dione), an analogcontaining no spacer arm between the 5-HT and the maleimide molecules(FIG. 19a), effectively inhibited the binding of ³H-5-HT to its high andlow affinity binding sites with IC₅₀ values of about 0.001 and 50 μM,respectively. LBT3002, an analog containing 3 carbon molecules in thespacer arm (FIG. 20a), inhibited the binding of ³H-5-HT to its high andlow affinity binding sites with IC₅₀ values of about 0.00003 and 200 μM,respectively. LBT3004, an analog containing 2 carbon molecules in thespacer arm (FIG. 21a), inhibited the binding of ³H-5-HT to its high andlow affinity binding sites with IC₅₀ values of about 0.2 and 50 μM,respectively. LBT3005, an analog containing cyclohexane carboxylicmolecule in the spacer arm (FIG. 22a), inhibited the binding of ³H-5-HTto its high and low affinity binding sites with IC₅₀ values of 0.5 and300 μM, respectively. These results clearly indicate irreversiblebinding drugs can be designed using the technology described.Additionally, the technology is applicable to many small molecules andcan be applied to further improve the efficacy of existing molecules.

[0228] The entire disclosures of all publications, patents and patentapplications referred to herein are hereby incorporated by reference.

[0229] Throughout the specification the aim has been to describe thepreferred embodiments of the invention without limiting the invention toany one embodiment or specific collection of features. Those of skill inthe art will appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the appendant claims.

1. A process for modifying a parent ligand, comprising attaching to saidparent ligand a conjugation agent that is reactive with a moiety of atarget receptor to which said parent ligand binds, wherein when saidparent ligand binds to the receptor a covalent bond is formed betweensaid conjugation agent and said moiety, and wherein the parent ligandbinds specifically with a nucleoside transporter.
 2. The process ofclaim 1 , wherein the conjugation agent is attached to the parent ligandthrough a spacer.
 3. The process of claim 2 , wherein the spacercomprises a group selected from the group consisting of alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, oxoalkyl, heterooxoalkyl,alkenyl, hetero alkenyl, aralkyl, hetero aralkyl, aryl and heteroarylradicals.
 4. The process of claim 1 , wherein the conjugation agent isselected from the group consisting of a sulfhydryl group specificconjugation agent, an amino group specific conjugation agent, a carboxylgroup specific conjugation agent, a tyrosine specific conjugation agent,an arginine specific conjugation agent, a histidine specific conjugationagent, a methionine specific conjugation agent, a tryptophan specificconjugation agent, and a serine specific conjugation agent.
 5. Theprocess of claim 1 , wherein the conjugation agent is a sulfhydryl groupspecific conjugation agent selected from the group consisting ofN-maleimide and N-maleimide derivatives.
 6. The process of claim 5 ,wherein the N-maleimide derivatives are selected from the groupconsisting of disulfide reagents including 5′-dithiobis-(2-nitrobenzoicacid), 4,4′-dithiodipyridine, methyl-3-nitro-2-pyridyl disulfide, andmethyl-2-pyridyl disulfide.
 7. The process of claim 1 , wherein theconjugation agent is selected from the group consisting of alkylatingagents and acylating agents.
 8. A process for modifying a parent ligand,comprising attaching to said parent ligand a sulfhydryl group specificconjugation agent that is reactive with a sulfhydryl group of a targetreceptor to which said parent ligand binds, wherein when said parentligand binds to the receptor a covalent bond is formed between saidconjugation agent and said sulfhydryl group, and wherein the parentligand binds specifically with a serotonin receptor.
 9. A modifiedligand produced by the process of claim 1 .
 10. A modified ligandproduced by the process of claim 8 .
 11. A modified ligand having thegeneral formula: L—R₁—A  (I) wherein L is a parent ligand that bindsspecifically with a target receptor comprising a nucleoside transporter;wherein A is a conjugation agent that is reactive with a moiety of thetarget receptor to which the parent ligand binds, such that when saidparent ligand binds to the receptor a covalent bond is formed betweensaid conjugation agent and said moiety; and R₁ is an optional spacer.12. The modified ligand of claim 11 , wherein the spacer comprises anon-hydrolysable radical selected from the group consisting of alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, oxoalkyl, heterooxoalkyl,alkenyl, hetero alkenyl, aralkyl, hetero aralkyl, aryl and heteroarylradicals.
 13. The modified ligand of claim 11 , wherein the conjugationagent is a sulfhydryl group specific conjugation agent.
 14. The modifiedligand of claim 12 , wherein the sulfhydryl group specific conjugationagent is selected from the group consisting of N-maleimide andN-maleimide derivatives.
 15. The modified ligand of claim 11 , whereinthe parent ligand binds specifically with an es nucleoside transporter.16. The modified ligand of claim 11 , which is interactive with esnucleoside transporter and/or nucleoside/nucleotide/nucleobase-sensitiveproteins, wherein said modified ligand has a general formula selectedfrom the group consisting of:

wherein A is N-maleimide, 2-pyridyldithio, or halogen; X is NH, S, or O;Y is H, halogen, NH₂, or O; Z is H, halogen, or CH₃; R₁ is spacer arm;and R₂ is H, β-D-ribose, β-D-2-deoxyribose, or their 5′-mono-, 5′ di-,and 5′ tri-phosphate.
 17. The modified ligand of claim 11 , which has ageneral formula selected from the group consisting of:

wherein R₄ is 4-[N-methyl]cyclohexane carboxylate, N-[m-benzoate],4-[p-phenyl]butyrate, N-[γ-butyrate], N-[α-acetate], orN-[ε-caproylate];

wherein R₄ is 4-[N-methyl]cyclohexane carboxylate, N-[m-benzoate],4-[p-phenyl]butyrate, N-[γ-butyrate], N-[α-acetate], orN-[ε-caproylate];

wherein R₅ is 4-carbonyl-α-methyl-α-toluene,6-[α-methyl-α-tuloamido]-hexanoate, N-[3-propionate], or6-[3′-propioamido]hexanoate; and

wherein R₅ is 4-carbonyl-α-methyl-α-toluene,6-[α-methyl-α-tuloamido]-hexanoate, N-[3-propionate], or 6-[3′-propioamido]hexanoate.
 18. A modified ligand having the generalformula: L—R₁—A  (I) wherein L is a parent ligand that bindsspecifically with a target serotonin receptor; wherein A is aconjugation agent that is reactive with a sulfhydryl group of saidtarget receptor to which the parent ligand binds, such that when saidparent ligand binds to the receptor a covalent bond is formed betweensaid conjugation agent and said sulfhydryl group; and R₁ is an optionalspacer.
 19. The modified ligand of claim 18 , wherein the parent ligandcomprises serotonin or a precursor or analog thereof.
 20. The modifiedligand of claim 18 , wherein the modified ligand has a structureselected from the group consisting of the structures shown below:

LBT3001 (1-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-dione)

LBT3002(4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-butyramide)

LBT3004(3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-propionamide);and

LBT3005 (4-(2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl)-cyclohexanecarboxylic acid [2-(5-hydroxy-lH-indol-3-yl)-ethyl]-amide)
 21. Acomposition comprising the modified ligand of claim 11 and apharmaceutically acceptable carrier.
 22. A composition comprising themodified ligand of claim 18 and a pharmaceutically acceptable carrier.23. A method of detecting the presence of a target receptor in a testsample, comprising: contacting said sample with the modified ligand ofclaim 11 , wherein said modified ligand binds said target receptor ifpresent in said test sample; and detecting the presence of a complexcomprising said modified ligand and said receptor in said contactedsample.
 24. A method of quantifying the presence of a target receptor ina test sample, comprising: contacting said sample with the modifiedligand of claim 11 , wherein said modified ligand binds said targetreceptor if present in said test sample; measuring the concentration ofa complex comprising said modified ligand and said receptor in saidcontacted sample; and relating said measured complex concentration tothe concentration of said receptor in said sample.
 25. A method ofdetecting the presence of a target receptor in a test sample,comprising: contacting said sample with the modified ligand of claim 18, wherein said modified ligand binds said target receptor if present insaid test sample; and detecting the presence of a complex comprisingsaid modified ligand and said receptor in said contacted sample.
 26. Amethod of quantifying the presence of a target receptor in a testsample, comprising: contacting said sample with the modified ligand ofclaim 18 , wherein said modified ligand binds said target receptor ifpresent in said test sample; measuring the concentration of a complexcomprising said modified ligand and said receptor in said contactedsample; and relating said measured complex concentration to theconcentration of said receptor in said sample.
 27. A probe thatcovalently binds to a target receptor, said probe comprising themodified ligand of claim 11 having a reporter molecule associatedtherewith.
 28. Use of the modified ligand of claim 11 or of the probe ofclaim 27 in the study of target receptor function.
 29. A probe thatcovalently binds to a target receptor, said probe comprising themodified ligand of claim 18 having a reporter molecule associatedtherewith.
 30. Use of the modified ligand of claim 18 or of the probe ofclaim 29 in the study of target receptor function.